Quantitation of methylated hemoglobin adducts in a signature peptide from rat blood by liquid chromatography/negative electrospray ionization tandem mass spectrometry

Hemoglobin adducts are often used as biomarkers for exposure to reactive chemicals in toxicology studies. Therefore, fast, sensitive, accurate, and reproducible methods for quantifying these protein adducts are key to evaluate test material dosimetry. A methodology has been developed for the quantitation of methylated hemoglobin adducts isolated from rats exposed to the model alkylating agent: methyl methane sulfonate (MMS). After 4 days of MMS exposure by oral gavage, hemoglobin was isolated from rat blood and digested with trypsin. The tryptic digestion solution was used for the adducted hemoglobin signature peptide quantitation via liquid chromatography/negative tandem mass spectrometry (LC/ESI-MS/MS). The limit of quantitation (LOQ) for the methylated hemoglobin beta chain N-terminal signature peptide (MeVHLTDAEK) was 1.95 ng/mL (5.9 pmol/mg globin). The calibration curves were linear over a concentration range of 1.95 to 625 ng/mL, with a correlation coefficient R2 >0.998, accuracy of 85.8 to 119.3%, and precision of 0.9 to 19.4%.     

A liquid chromatography tandem mass spectrometric method for in vivo dose monitoring of diepoxybutane, a metabolite of butadiene

1,3-Butadiene, a common air pollutant formed in the combustion of organic matter, has been assessed by the U.S. EPA to be a strongly carcinogenic compound. This risk assessment is very uncertain because of the lack of information on the dose of the powerful carcinogenic metabolite diepoxybutane (DEB). This report presents an analytical method for in vivo dose monitoring of a unique marker for DEB. For a large number of alkylating agents in vivo doses are monitored by measurement by gas chromatography/mass spectrometry (GC/MS) of adducts to N-terminal valine in hemoglobin (Hb), using a modified Edman degradation method. This method is applicable to monofunctional epoxides from butadiene. However, in reaction with N-terminal valine, DEB forms an adduct which is ring-closed to a pyrrolidine, N,N-(2,3-dihydroxy-1,4-butadiyl)valine, with a tertiary amino group that prevents detachment of the alkylated valine by the Edman reagent. Therefore a method has been developed based on the analysis by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) of the N-modified N-terminal peptides enriched after trypsin digestion of globin. In this study Hb samples from mice injected intraperitoneally with (+/-)-DEB were examined qualitatively and quantitatively with regard to the ring-closed adduct. The N-terminal pyrrolidine-heptapeptide was identified in treated mice. The highest adduct levels were obtained in samples from animals given the highest dose of DEB and the adduct levels were below the detection level in control mice.     

Formation of O6,7-dimethylguanine residues in calf thymus deoxyribonucleic acid treated with carcinogenic N-methyl-N-nitrosourea in vitro

Treatment of calf thymus deoxyribonucleic acid (DNA) in vitro with a methylating carcinogen, N-methyl-N-nitrosourea (MNU), in phosphate buffer (pH 7.2) resulted in formation of O6,7-dimethylguanine residues in DNA besides the well-known methylated DNA adducts, 7-methylguanine, O6-methylguanine and 3-methyladenine. The product ratio (%) of O6,7-dimethylguanine versus 7-methylguanine was 0.32 after one MNU treatment. The significance of formation of O6,7-dimethylguanine residues in DNA is discussed briefly in relation to the carcinogenicity of MNU.     

Structural analysis of naphthoquinone protein adducts with liquid chromatography/tandem mass spectrometry and the scoring algorithm for spectral analysis (SALSA)

The relative reactivities of various naphthoquinone isomers (1,4-, 1,2- and 2-methyl-1,4-naphthoquinone) to two test proteins, apomyoglobin and human hemoglobin, were evaluated via liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). The structural characterization of the resulting adducts was also obtained by LC/ESI-MS analysis of the intact proteins. The reactive sites of apomyoglobin and human hemoglobin with 1,4-naphthoquinone and 1,2-naphthoquinone were also identified through characterization of adducted tryptic peptides by use of high-pressure liquid chromatography/electrospray ionization with tandem mass spectrometry (HPLC/ESI-MS/MS), TurboSEQUEST, and the scoring algorithm for spectral analysis (SALSA). Four adducted peptides, which were formed by nucleophilic addition of a lysine amino acid residue to 1,4-naphthoquinone, were also identified, as was an adducted peptide from incubation of 1,2-naphthoquinone with apomyoglobin. In the case of incubation of human hemoglobin with the two naphthoquinones, two adducted peptides were identified from the N-terminal valine modification of the alpha and beta chains of human hemoglobin. The adducted protein formation may imply that naphthalene produces its in vivo toxicity through 1,2- and 1,4-naphthoquinone metabolites reacting with biomolecular proteins.     

Alteration in the choice of DNA repair pathway with increasing sequence selective DNA alkylation in the minor groove

BACKGROUND: Many conventional DNA alkylating anticancer drugs form adducts in the major groove of DNA. These are known to be chiefly repaired by both nucleotide (NER) and base (BER) excision repair in eukaryotic cells. Much less is known about the repair pathways acting on sequence specific minor groove purine adducts, which result from a promising new class of anti-tumour agents. RESULTS: Benzoic acid mustards (BAMs) tethering 1-3 pyrrole units (compounds 1, 2 and 3) show increasing DNA sequence selectivity for alkylation from BAM and 1, alkylating primarily at guanine-N7 in the major groove, to 3 which is selective for alkylation in the minor groove at purine-N3 in the sequence 5'-TTTTGPu (Pu=guanine or adenine). This increasing sequence selectivity is reflected in increased toxicity in human cells. In the yeast Saccharomyces cerevisiae, the repair of untargeted DNA adducts produced by BAM, 1 and 2 depends upon both the NER and BER pathways. In contrast, the repair of the sequence specific minor groove adducts of 3 does not involve known BER or NER activities. In addition, neither recombination nor mismatch repair are involved. Two disruptants from the RAD6 mutagenesis defective epistasis group (rad6 and rad18), however, showed increased sensitivity to 3. In particular, the rad18 mutant was over three orders of magnitude more sensitive to 3 compared to its isogenic parent, and 3 was highly mutagenic in the absence of RAD18. Elimination of the sequence specific DNA adducts formed by 3 was observed in the wild type strain, but these lesions persisted in the rad18 mutant. CONCLUSIONS: We have demonstrated that the repair of DNA adducts produced by the highly sequence specific minor groove alkylating agent 3 involves an error free adduct elimination pathway dependent on the Rad18 protein. This represents the first systematic analysis of the cellular pathways which modulate sensitivity to this new class of DNA sequence specific drugs, and indicates that the enhanced cytotoxicity of certain sequence specific minor groove adducts in DNA is the result of evasion of the common excision repair pathways.     

A practical approach for the chemical synthesis of 2′-deoxyguanosine-C8 adducts with mutagenic/carcinogenic amino- or nitro-arenes

Synthetic methods for the preparation of 2′-deoxyguanosine-C8 (dG-C8) adducts with several mutagenic and carcinogenic amino- or nitro-arenes were developed using the palladium-mediated cross-coupling reaction of protected 8-amino-dG with bromoarenes in around 80% yields, followed by conventional deprotection procedures. This approach can be applied to preparation of a variety of authentic dG-C8 adducts with amino or nitro-arenes.     

Optimization of a biomimetic transamination reaction

For a range of protein substrates, N-terminal transamination offers a convenient way to install a reactive ketone or aldehyde functional group at a single location. We report herein the effects of the identity of N-terminal residues on the product distribution generated upon reaction with pyridoxal 5'-phosphate (PLP). This study was accomplished through the combination of solid-phase peptide synthesis with detailed liquid chromatography-mass spectrometry analysis. Many N-terminal amino acids provided high yields of the desired transaminated products, but some residues (His, Trp, Lys, and Pro) generated adducts with PLP itself. N-terminal Cys and Ser residues were observed to undergo beta-elimination in addition to transamination, and the transamination product of N-terminal Gln was resistant to subsequent oxime formation attempts. The information generated through the screening of peptide substrates was successfully applied to a protein target, changing an initially unreactive terminus into one that could be modified in high (70%) yield. Thus, these studies have increased our predictive power for the reaction, both in terms of improving conversion and suppressing reaction byproducts. An initial set of guidelines that may be used to increase the applicability of this reaction to specific proteins of interest is provided.     

Quantitation of lower levels of the DNA adduct of thymidylyl(3'-5')thymidine methyl phosphotriester by liquid chromatography/negative atmospheric pressure chemical ionization tandem mass spectrometry

A sensitive method has been developed for the direct quantitation of the methyl phosphotriester DNA adduct of thymidyl(3'-5')thymidine (dTp(Me)dT) from enzymatic DNA hydrolysates prepared from cultured cells treated with low levels of N-methyl-N-nitrosourea (MNU) and methyl methane sulfonate (MMS), by rapid and selective liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry (LC/APCI-MS/MS). The lower limit of quantitation was 0.1 ng/mL (6.4 adducts per 10(8) nucleotides). Linearity of the calibration curve was greater than 0.999 from 0.1 to 50 ng/mL. Intra-day precision for three levels of quality control samples ranged from 4.27 to 15.62%. Interday precision ranged from 2.46 to 11.95%. Using this method, the levels of dTp(Me)dT in DNA enzymatic hydrolysates obtained from a series of incubations of mouse lymphoma cells with low doses of MNU (50 microM) were quantified.     

Identification of protein adduction using mass spectrometry: Protein adducts as biomarkers and predictors of toxicity mechanisms

The determination of protein‐xenobiotic adducts using mass spectrometry is an emerging area which allows detailed understanding of the underlying mechanisms involved in toxicity. These approaches can also be used to reveal potential biomarkers of exposure or toxic response. The following review covers studies of protein adducts resulting from exposure to a wide variety of xenobiotics including organophosphates, polycyclic aromatic hydrocarbons, acetaminophen, alkylating agents and other related compounds. Copyright © 2016 John Wiley & Sons, Ltd.     

Homocysteine thiolactone and protein homocysteinylation: mechanistic studies with model peptides and proteins

In vitro incubations were performed to show that homocysteine thiolactone could generate covalent adducts with model peptides and proteins. MS and MS/MS data suggest that the thiolactone reacts with the side-chain amino group of lysine residues as well as with the N-terminal amino group or C-terminal carboxy group. For larger peptides and proteins, the contribution from the in-amino groups of lysine residues should be predominant. These data could help explain the detrimental effects of elevated levels of homocysteine and homocysteine thiolactone.     

Separation and enrichment of alkylated globin chains as a means of improving the sensitivity of hemoglobin adduct measurements

Measurement of adducts of hemoglobin is a reliable and quantitative method for monitoring exposure to genotoxic chemicals. To make the approach applicable to the low levels of adducts originating from exposure to chemicals in the environment, increased sensitivity of the analytical procedures is required. The method presented here is based on quantitative determination of low levels of adducts after purification and enrichment of chemically modified (adducted) globin chains on CM-Sepharose CL-6B. In the developmental work, human globin was used after alkylation by radiolabelled ethylene oxide, styrene oxide or N-ethyl-N-nitrosourea. Ethylene oxide reacts mainly with the amino terminal valine and nitrogens in the imidazole ring of histidine, while N-ethyl-N-nitrosourea has a particularly high reactivity towards free carboxy groups of acidic amino acids. Globin chains with adducts to the carboxy groups were especially easy to separate from the non-modified chains. Ethyl adducts to carboxy groups in hemoglobin were shown to be sufficiently stable in vivo to be used for dose monitoring.     

Novel lipid hydroperoxide-derived hemoglobin histidine adducts as biomarkers of oxidative stress

Hemoglobin (Hb) adducts have long been used as dosimeters of exposure to xenobiotics and endogenously formed reactive metabolites. In this study, hemoglobin chains were separated from each other and their prosthetic heme groups and reacted with 4-oxo-2-nonenal, a major breakdown product of lipid hydroperoxides. The adducts were characterized by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-TOF/MS) analysis of the intact proteins and by a combination of liquid chromatography/electrospray ionization/tandem MS (MS/MS) and MALDI-TOF/MS/MS analysis of the tryptic peptides. Covalent modifications were found on both hemoglobin chains. The location was determined to be on H20 of the alpha-hemoglobin chain and on H(63) of the beta-hemoglobin chain. Molecular modeling revealed that these two residues were two most solvent accessible H residues present in intact Hb. The proposed reaction mechanism is based on that described for the reaction of 4-hydroxy-2-nonenal with proteins. Initial nucleophilic Michael addition is followed by hydration of the resulting aldehyde, cyclization, and two sequential dehydration reactions to give stable furan derivatives. This results in the addition of 136 Da from 4-oxo-2-nonenal to give adducts corresponding to (17)VGAH(.) AGEYGAEALER(31) from alpha-hemoglobin and (62)AH(.) GK(65) from beta-hemoglobin. These hemoglobin modifications can potentially serve as biomarkers of lipid hydroperoxide-mediated macromolecule damage and may reflect an indirect measurement of the potential for DNA damage in vivo.     

V-type nerve agents phosphonylate ubiquitin at biologically relevant lysine residues and induce intramolecular cyclization by an isopeptide bond

Toxic organophosphorus compounds (e.g., pesticides and nerve agents) are known to react with nucleophilic side chains of different amino acids (phosphylation), thus forming adducts with endogenous proteins. Most often binding to serine, tyrosine, or threonine residues is described as being of relevance for toxicological effects (e.g., acetylcholinesterase and neuropathy target esterase) or as biomarkers for post-exposure analysis (verification, e.g., albumin and butyrylcholinesterase). Accordingly, identification of novel protein targets might be beneficial for a better understanding of the toxicology of these compounds, revealing new bioanalytical verification tools, and improving knowledge on chemical reactivity. In the present study, we investigated the reaction of ubiquitin (Ub) with the V-type nerve agents Chinese VX, Russian VX, and VX in vitro. Ub is a ubiquitous protein with a mass of 8564.8 Da present in the extra- and intracellular space that plays an important physiological role in several essential processes (e.g., proteasomal degradation, DNA repair, protein turnover, and endocytosis). Reaction products were analyzed by matrix-assisted laser desorption/ionization-time-of-flight- mass spectrometry (MALDI-TOF MS) and μ-high-performance liquid chromatography online coupled to UV-detection and electrospray ionization MS (μHPLC-UV/ESI MS). Our results originally document that a complex mixture of at least mono-, di, and triphosphonylated Ub adducts was produced. Surprisingly, peptide mass fingerprint analysis in combination with MALDI and ESI MS/MS revealed that phosphonylation occurred with high selectivity in at least 6 of 7 surface-exposed lysine residues that are essential for the biological function of Ub. These reaction products were found not to age. In addition, we herein report for the first time that phosphonylation induced intramolecular cyclization by formation of an isopeptide bond between the ε-amino group of a formerly phosphonylated lysine and the side chain of an adjacent acidic glutamic acid residue.

Adducts of N-terminal valines in hemoglobin with isoprene diepoxide, a metabolite of isoprene

Isoprene (2-methylbuta-1,3-diene) is a multi-site carcinogen in rodents. To evaluate the role of the diepoxide metabolite (1,2:3,4-diepoxy-2-methylbutane) in carcinogenesis, measurements of in vivo doses of the diepoxide are needed. The in vivo dose may be inferred from levels of reaction products with hemoglobin (Hb adducts). This report presents in vitro studies of the adduct formation by the diepoxide of isoprene with valinamide and oligopeptides as model compounds of N-terminal valines in hemoglobin (Hb). In the reaction with valinamide it was shown that isoprene diepoxide forms as the main product a ring-closed adduct, which is a pyrrolidine derivative [N,N-(2,3-dihydroxy-2-methyl-1,4-butadiyl)valinamide, MPyr-Val]. The analysis was performed by gas chromatography/mass spectrometry (GC/MS) (EI and PICI) after acetylation. The ring-closed adduct was also identified by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) as the main product in the reaction between isoprene diepoxide and standard hepta- or (2H8)octapeptides, corresponding to the N-terminal peptides of the alpha-chains in mouse and rat Hb. These peptides, alkylated with isoprene diepoxide, to be used as internal standards and calibration standards for quantification of MPyr-adduct levels in vitro and in vivo, were analyzed with respect to the degree of MPyr-alkylation by two independent methods, amino acid analysis and HPLC-UV; similar results were obtained using these methods. A method for measurement of Hb adducts as modified peptides, used earlier to measure a similar adduct to N-terminal valines in Hb from the diepoxide of 1,3-butadiene, has in the present work been tested for application to isoprene diepoxide. The method is based on tryptic degradation of globin and LC/ESI-MS analysis of N-terminal Pyr-heptapeptides of the Hb alpha-chain enriched by HPLC. MPyr-adduct levels in isoprene diepoxide alkylated hemolysate from mouse erythrocytes incubated with different concentrations of isoprene diepoxide (2 and 10 mM) for 1 h were quantified. The adduct level was about 50 nmol/g alpha-chain Hb per mM x h. From the adduct levels the rate constant of isoprene diepoxide for reaction with N-terminal valine was calculated to be about 1.6 times faster than for diepoxybutane.     

A new deuterated alkylating agent for quantitative proteomics

Weakly basic molecules containing a double bond, such as 2- and 4-vinylpyridine, are able to react and selectively alkylate -SH groups in proteins, thus preventing their re-oxidation to disulphide bridges. In contrast to conventional alkylating agents such as iodoacetamide and non-charged acrylamide derivatives, such molecules achieve 100% alkylation of all -SH residues, even in complex proteins, without reacting with other functional groups. Their use is particularly effective in proteome analysis and more generally for analyzing proteins in which the -SH groups should be blocked. Additionally, the use of vinylpyridines, partially or totally deuterated and thus with a mass difference compared with their non-deuterated counterparts of 4-7 Da, allows studies of induction/repression of protein synthesis (quantitative proteomics).     

A method to enhance a1 ions and application for peptide sequencing and protein identification

A simple and effective method was developed for peptide sequencing and protein identification through the determination of its N-terminal residue. The method of N-terminal carbamidomethylation with iodoacetamide could specifically and remarkably enhance the intensity of a₁ ions in the tandem mass spectra of the peptide derivatives without significantly altering their fragmentation pattern, thus allowing determination of their N-terminal residues. The effectiveness and specificity of the method was demonstrated by confirming and extending sequence interpretation of several model peptides and proteins. The developed method was then applied in the LC-MS/MS analysis of the tryptic digests of myoglobin and a whole protein extract from rat heart tissues. The results from database searches were well validated with the enhancement of a₁ ions in tandem mass spectra and the specificity of protein identification was obtained when the information of N-terminal residues was included in the database search.     

Cysteine adducts of human haemoglobin measured by isoelectric focusing in polyacrylamide gels with a non-linear pH gradient

The in vitro formation of adducts from human haemoglobin formed by alkylation with methyl-methanesulphonate, dimethyl sulphate and iodoacetamide was determined with isoelectric focusing in ultra-thin polyacrylamide gels with a non-linear pH gradient. The most important adduct seen in the gels was identified as HbA alkylated at the beta-93 cysteine. Influences of the chemical nature of the alkylating agents and of the biological environment are discussed. The method is suggested to be applicable to monitoring the biological effects of low, long-term exposure to mixtures of alkylating agents or of exposure to unknown compounds.     

Methylating peptides to prevent adduct ion formation also directs cleavage in collision-induced dissociation mass spectrometry

We investigated the effect of N-terminal amino group and carboxyl group methylation on peptide analysis by electrospray mass spectrometry (ESI-MS) and tandem mass spectrometry (ESI-MS/MS). Permethylation of the N-terminal amino group and the carboxyl groups can reduce metal ion adducts but does not enhance sensitivity in electrospray as previously observed for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. N-terminal trimethylated peptides exhibit collision-induced dissociation (CID) tandem mass spectra that differ from their unmodified analogs; the results support the mobile proton hypothesis of peptide fragmentation. A permanent positive charge at the N-terminus leads to competition between permanent-charge directed processes and loss of the N-terminal trimethyl amino group. Carboxyl methylation has no effect on fragmentation behavior other than to shift the mass of fragments containing methylated carboxyl groups. Comparison of regular and tandem mass spectra of different methylated peptides allowed probing the location of incomplete methylation, the proton displaced by alkali metal ions and the purity of a mass-selected methylated peptide ion.     

The reaction of 4-(p-nitrobenzyl)pyridine with some electrophiles

The nucleophile 4-(p-nitrobenzyl)pyridine was allowed to react with four carcinogenic alkylating agents, chloromethyl methyl ether, bis(chloromethyl) ether, glycol sulfate and propane sultone, one carcinogenic acylating agent, N,N-dimethylcarbamyl chloride, and one noncarcinogenic electrophile, perchlorocyclobutenone. The structures of the major products formed, which are substituted pyridinium salts or 1,4-dihydropyridines, were determined.     

Alkylation of heme by artemisinin,an antimalarial drug

The reductive activation of artemisinin (or artemether) by ferroprotoporphyrin-IX, the prosthetic group of hemoglobin, is able to produce covalent adducts heme-artemisinin in high yield under very mild conditions. This adduct formation, using the natural target of an endoperoxide antimalarial drug, confirms the alkylating ability of this class of antimalarial drugs, which has already been reported when using a synthetic manganese porphyrin.